A rapid method (7 sec.) of separation of mitochondria using digitonin was compared to mitochondria separated from rapidly frozen (millisec) hepatocytes, fractionated in nonaqueous media. Comparison of the results shows that the slower digitonin fractionation allows redistribution of Alpha-oxoglutarate and inorganic phosphate. Measurement of other amino acids and metabolites agree suggesting extensive metabolite binding within mitochondrial matrix. This conclusion is substantiated by the apparent disequilibrium of calculated oxaloacetate values with the MDH and GOT reaction in mitochondrial, but not cytoplasmic space. Total cellular levels of adenine, granine and uridene nucleotides fail to show the expected equilibrium reactions with the adenylate kinase, nucleoside diphosphate kinase and nucleoside monophosphate kinase. While the explanation of this anomoly is not clear, it, in part, may be due to artifactual decomposition of tri and di phosphonucleotides to the monophosphate nucleotides during tissue processing. There is a significant and unexpected increase Rho/K+ ion within the mitochondrial matrix in comparison with cytoplasm. The reason for this phenomena is being investigated.